Reporter gene system for use in cell-based assessment of inhibitors of the Hepatitis C virus protease

Abstract

A cell-based assay system in which the detection of the reporter gene activity, or secreted alkaline phosphatase (SEAP), is dependent upon the protease activity of the Hepatitis C virus NS3 gene product. This system can be used to assess the activity of candidate protease inhibitors in a mammalian cell-based assay system. The assay system is simpler than previously described assays due to the use of SEAP which allows the reporter gene activity to be quantified by measuring the amount of secreted gene product in the cell media by monitoring the conversion of luminescent or calorimetric alkaline phosphatase substrate.

We claim:  
     
       1. A reporter gene system useful to assess compounds which augment or inhibit the activity of Hepatitis C virus NS3 protease, comprising: 
       a) a recombinant viral vector comprising a DNA molecule which encodes an RNA polymerase compatible with said viral vector; and  
       b) a recombinant plasmid comprising a DNA molecule comprising a Hepatitis C virus/secreted alkaline phosphatase (HCV/SEAP) gene construct operably linked to a promoter, said promoter being compatible with said RNA polymerase, wherein upon co-transfection into a host cell with said recombinant viral vector, said Hepatitis C virus/SEAP gene construct is under the transcriptional control of said promoter, and wherein said RNA polymerase is acting in trans;  
       and wherein a presence of SEAP activity is indicative of Hepatitis C virus NS3 protease activity. 
     
     
       2. The reporter gene system of claim  1 , wherein said recombinant plasmid is a pTM3 plasmid. 
     
     
       3. The reporter gene system of claim  1 , wherein said promoter is a T7 RNA polymerase promoter. 
     
     
       4. The reporter gene system of claim  1 , wherein said promoter is a vaccinia virus compatible promoter. 
     
     
       5. The reporter gene system of claim  1 , wherein said recombinant viral vector comprises a promoter selected from the group consisting of a Simian Virus (SV40) promoter, Rous Sarcoma Virus (RSV) promoter, Adenovirus (ADV) promoter, and Bovine Papilloma Virus (BPV) promoter. 
     
     
       6. The reporter gene system of claim  1 , wherein said recombinant viral vector and said recombinant plasmid are capable of being transfected into a target mammalian cell line selected from the group consisting of HeLa cells, Chinese Hamster Ovary cells, CV1 African Green Monkey cells, BSC 1 cells, and Baby Hamster Kidney cells. 
     
     
       7. A reporter gene system useful to assess compounds which augment or inhibit the activity of Hepatitis C virus NS3 protease comprising: 
       a) a first recombinant viral vector comprising a DNA molecule which encodes an RNA polymerase compatible with said viral vector; and  
       b) a second recombinant viral vector comprising a DNA molecule which encodes the Hepatitis C virus/SEAP gene construct operably linked to a promoter, said promoter being compatible with said RNA polymerase, and wherein upon co-transfection of said first and second recombinant viral vectors into a host cell, said HCV/SEAP gene construct is under the transcriptional control of said promoter, and wherein said RNA polymerase is acting in trans;  
       and wherein a presence of SEAP activity is indicative of Hepatitis C virus NS3 protease activity. 
     
     
       8. The reporter gene system of claim  7  wherein said second recombinant viral vector is a vHCAP1 vector. 
     
     
       9. The reporter gene system of claim  8  wherein said vHCAP1 vector comprises a NS2 protease gene, a NS3 protease gene, and a SEAP gene linked in a cis configuration as depicted in SEQ ID No. 1. 
     
     
       10. The reporter gene system of claim  7 , wherein said second recombinant viral vector is a vHCAP3 vector. 
     
     
       11. The reporter gene system of claim  10  wherein said vHCAP3 vector comprises a NS2 protease gene, a mutant NS3 protease gene, and a SEAP gene linked in a cis configuration as depicted in SEQ ID No. 9. 
     
     
       12. The reporter gene system of claim  7  wherein said second recombinant viral vector is a vHCAP4 vector. 
     
     
       13. The reporter gene system of claim  12 , wherein said vHCAP4 vector comprises a NS2 protease gene, a mutant NS3 protease gene, and a SEAP gene linked in a cis configuration as depicted in SEQ ID No. 16. 
     
     
       14. The reporter gene system of claim  7  wherein said promoter is a T7 RNA polymerase promoter. 
     
     
       15. The reporter gene system of claim  7  wherein said promoter is a vaccinia virus compatible promoter. 
     
     
       16. The reporter gene system of claim  7  wherein said recombinant viral vector comprises a promoter selected from the group consisting of a Simian Virus (SV40) promoter, Rous Sarcoma Virus (RSV) promoter, Adenovirus (ADV) promoter, and Bovine Papilloma Virus (BPV) promoter. 
     
     
       17. The reporter gene system of claim  3 , wherein said recombinant viral vector and said recombinant plasmid are capable of being transfected into a target mammalian cell line selected from the group consisting of HeLa cells, Chinese Hamster Ovary cells, CV1 African Green Monkey cells, BSC 1 cells, and Baby Hamster Kidney cells. 
     
     
       18. A method of assessing compounds which augment or inhibit the activity of Hepatitis C virus NS3 protease comprising 
       (a) incubating for 24 hours in a suitable growth medium in the presence or absence of a pharmacologically effective concentration of candidate compounds:  
       i) a control target mammalian cell line;  
       ii) a first target mammalian cell line which expresses a vHCAP1 vector, said vHCAP1 vector comprising a Hepatitis C virus/SEAP gene construct;  
       iii) a second target mammalian cell line which expresses a vHCAP4 vector, said vHCAP4 vector comprising a Hepatitis C virus/SEAP gene construct; and  
       iv) a third target mammalian cell line which expresses a recombinant viral vector comprising a DNA molecule which encodes an RNA polymerase operably linked to a promoter;  
       (b) measuring the amount of SEAP activity secreted from said cell lines; and  
       (c) determining whether said candidate compounds augmented or inhibited Hepatitis C virus NS3 protease by comparing the SEAP activity of said control, first, second, and third target cell lines.  
     
     
       19. A method of assessing compounds which augment or inhibit the activity of Hepatitis C virus NS3 protease comprising 
       (a) incubating for 24 hours in a suitable growth medium in the presence or absence of pharmacologically effective concentration of candidate compounds:  
       i) a control target mammalian cell line;  
       ii) a first target mammalian cell line which expresses a vHCAP3 vector, said vHCAP3 vector comprising a Hepatitis C virus/SEAP gene construct;  
       iii) a second target mammalian cell line which expresses a vHCAP4 vector, said vHCAP4 vector comprising a Hepatitis C virus/SEAP gene construct; and  
       iv) a third target mammalian cell line which expresses a recombinant viral vector comprising a DNA molecule which encodes an RNA polymerase operably linked to a promoter;  
       (b) measuring the amount of SEAP activity secreted from said cell lines; and  
       (c) determining whether said candidate compounds augmented or inhibited Hepatitis C virus NS3 protease by comparing the SEAP activity of said control, first, second, and third target cell lines.  
     
     
       20. A process for constructing a reporter gene system useful in the assessment of compounds which augment or inhibit the activity of Hepatitis C virus NS3 protease comprising: 
       (a) providing a recombinant viral vector comprising a DNA molecule encoding an RNA polymerase operably linked to a promoter, wherein said promoter is compatible with said viral vector, and wherein said RNA polymerase is expressed upon infection of a target mammalian cell line;  
       (b) providing a recombinant plasmid comprising a Hepatitis C virus/SEAP reporter gene construct, wherein said reporter gene construct comprises the NS2-NS3-NS4A-NS4B′-NS5A cleavage site-SEAP gene; and  
       (c) incubating said target mammalian cell line first with said recombinant viral vector, and then with said recombinant plasmid such that the DNA molecule encoding the Hepetitis C virus/SEAP reporter gene construct is under the transcriptional control of said promoter, wherein said RNA polymerase is acting in trans, and wherein said SEAP reporter gene is expressed and secreted from said target mammalian cell.